An approach to locate phosphorylation sites in a phosphoprotein: mass mapping by combining specific enzymatic degradation with matrix-assisted laser desorption/ionization mass spectrometry

Anal Biochem. 1994 May 15;219(1):9-20. doi: 10.1006/abio.1994.1224.


A rapid, picomole-scale method is described to locate phosphorylation sites in phosphoproteins by using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) combined with enzymatic modification of the analyte. There are three steps to locate phosphorylation sites in a phosphoprotein: (i) degradation of the phosphoprotein into small peptides by specific enzymatic or chemical reactions; (ii) identification of the phosphopeptides by -80 (or multiples of -80)-Da mass shifts in the mass spectra after dephosphorylation with alkaline phosphatase; (iii) location of the phosphorylation sites by mass mapping. As the size of the protein increases, it is advantageous to fractionate the mixture by HPLC and analyze each fraction by MALDI-TOF-MS. To perform mass mapping, the primary structure of the protein must be known. Bovine beta-casein was analyzed by this method. The conclusions about the specific phosphorylation sites of bovine beta-casein from our data coincide with previously reported results. From calculations, it is found that a mass spectrometer with 0.1% mass accuracy is sufficient, for mass mapping, to identify completely or partially digested tryptic peptides in the mass range of 100-8000 Da from bovine beta-casein (MW 23,983).

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Caseins / chemistry*
  • Cattle
  • Chromatography, High Pressure Liquid / methods
  • Lasers
  • Mass Spectrometry / methods*
  • Molecular Sequence Data
  • Peptide Fragments / chemistry*
  • Peptide Fragments / isolation & purification
  • Peptide Mapping
  • Phosphopeptides / chemical synthesis
  • Phosphopeptides / chemistry*
  • Phosphoproteins / chemistry*
  • Phosphoproteins / metabolism
  • Phosphorylation
  • Sensitivity and Specificity


  • Caseins
  • Peptide Fragments
  • Phosphopeptides
  • Phosphoproteins